ctcf regulates ataxin-7 expression through promotion of a

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Neuron

Article

CTCF Regulates Ataxin-7 Expressionthrough Promotion of a ConvergentlyTranscribed, Antisense Noncoding RNABryce L. Sopher,1,11 Paula D. Ladd,4,11 Victor V. Pineda,1,11 Randell T. Libby,1 Susan M. Sunkin,9 James B. Hurley,2

Cortlandt P. Thienes,1 Terry Gaasterland,6,7 Galina N. Filippova,10 and Albert R. La Spada3,4,5,7,8,*1Department of Laboratory Medicine2Department of Biochemistry3Department of Medicine

University of Washington, Seattle, WA 98195, USA4Department of Pediatrics5Department of Cellular & Molecular Medicine6Scripps Institute for Oceanography7Institute for Genomic Medicine

University of California, San Diego, La Jolla, CA 92093, USA8Rady Children’s Hospital, San Diego, CA 92123, USA9Allen Institute for Brain Science, Seattle, WA 98103, USA10Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA11These authors contributed equally to this work

*Correspondence: [email protected] 10.1016/j.neuron.2011.05.027

SUMMARY

Spinocerebellar ataxia type 7 (SCA7) is a neurode-generative disorder caused by CAG/polyglutaminerepeat expansions in the ataxin-7 gene. Ataxin-7is a component of two different transcription coacti-vator complexes, and recent work indicates thatdisease protein normal function is altered in polyglut-amine neurodegeneration. Given this, we studiedhow ataxin-7 gene expression is regulated. Theataxin-7 repeat and translation start site are flankedby binding sites for CTCF, a highly conserved multi-functional transcription regulator. When we analyzedthis region, we discovered an adjacent alternativepromoter and a convergently transcribed antisensenoncoding RNA, SCAANT1. To understand howCTCF regulates ataxin-7 gene expression, we intro-duced ataxin-7 mini-genes into mice, and foundthat CTCF is required for SCAANT1 expression.Loss of SCAANT1 derepressed ataxin-7 sense tran-scription in a cis-dependent fashion and was accom-panied by chromatin remodeling. Discovery of thispathway underscores the importance of alteredepigenetic regulation for disease pathology at repeatloci exhibiting bidirectional transcription.

INTRODUCTION

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurological

disorder characterized by cerebellar and retinal degeneration

(Martin et al., 1994). SCA7 is caused by a CAG/polyglutamine

(polyQ) repeat expansion in the ataxin-7 gene and is therefore

one of nine polyQ neurodegenerative disorders (La Spada and

Taylor, 2010). Included in the CAG/polyQ repeat disease cate-

gory are spinobulbar muscular atrophy (SBMA), Huntington’s

disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA),

and five other forms of spinocerebellar ataxia (SCA1, 2, 3, 6,

and 17). Numerous lines of investigation in the polyQ disease

field suggest that expansion of the glutamine tract is a gain-of-

function mutation, and that the initiating event in disease patho-

genesis is transition of the polyQ expansion tract to an altered

conformation (Paulson et al., 2000; Ross, 1997). However, as

each polyQ disease displays distinct patterns of neuropathology

despite overlapping patterns of disease gene expression, it is

likely that the normal function, activities, and interactions of the

polyQ disease protein determine the cell-type specificity in

each disorder (La Spada and Taylor, 2003). Ataxin-7, the causal

protein in SCA7, contains a polyQ tract that ranges in size from

4–35 glutamines in normal individuals, but expands to 37–>400

glutamines in affected patients (David et al., 1997; Stevanin

et al., 2000). The glutamine tract is located in the amino-terminus

of ataxin-7, beginning at position #30. SCA7 is unique among

the CAG/polyQ repeat diseases, as patients with this disorder

develop a retinal degeneration phenotype, classified as a

cone-rod dystrophy (Ahn et al., 2005; To et al., 1993). To under-

stand the basis of SCA7 retinal degeneration and the reason for

the selective loss of photoreceptor cells in this disease, we, and

others, produced transgenic mice that recapitulated the

SCA7 cone-rod dystrophy phenotype and found that SCA7

retinal degeneration results from altered transcription regulation

(Helmlinger et al., 2006; La Spada et al., 2001; Yoo et al., 2003).

As the vast majority of CAG/polyQ disease proteins are well-

known transcription factors or can function as transcription

Neuron 70, 1071–1084, June 23, 2011 ª2011 Elsevier Inc. 1071

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Neuron

CTCF Promotes AS ncRNA to Control atx7 Expression

co-regulators (Riley and Orr, 2006), a role for transcription dysre-

gulation in SCA7 is consistent with an emerging view of these

disorders as ‘‘transcriptionopathies’’ (La Spada and Taylor,

2003). The existence of an interaction between ataxin-7 and

a retinal transcription factor, known as CRX, suggested that

ataxin-7 is a transcription factor (La Spada et al., 2001), and

this was supported by demonstration of a functional nuclear

localization signal in ataxin-7 (Chen et al., 2004). When studies

of the yeast ortholog of ataxin-7, Sgf73, indicated that Sgf73 is

part of the SAGA complex (Sanders et al., 2002), we, and others,

found that ataxin-7 is a core component of the analogous coac-

tivator complex in mammals, known as the STAGA (Spt3-Taf9-

Ada-Gcn5-acetyltransferase) complex (Helmlinger et al., 2004;

Palhan et al., 2005). STAGA is a transcriptional coactivator

complex with histone acetyltransferase (HAT) activity (Martinez

et al., 2001). In addition to being part of the STAGA complex,

yeast Sgf73 and mammalian ataxin-7 are respectively compo-

nents of the Ubp8/USP22 deubiquitination complex (Kohler

et al., 2008; Zhao et al., 2008). While the role of altered STAGA

and USP22 deubiquitination complex function in SCA7 disease

pathogenesis is unclear, recent studies of the related polyQ

disorder SCA1 indicate that the polyQ expansion in ataxin-1

attenuates the formation and function of the Capicua transcrip-

tion factor complex, contributing to SCA1 disease pathogenesis

through a partial loss-of-function mechanism (Chen et al., 2003;

Lim et al., 2008). Hence, polyQ disease may result from an alter-

ation of normal function, combined with a gain-of-function

mechanism, and in the case of SCA7, the native protein function

of ataxin-7 appears critically important for chromatin remodeling

at the level of histone acetylation and deubiquitination.

In addition to the CAG/polyQ repeat diseases, at least three

other subclasses of repeat expansion disease are recognized:

loss-of-function repeat expansion diseases, RNA gain-of-func-

tion repeat disorders, and polyalanine gain-of-function repeat

expansion diseases (La Spada and Taylor, 2010). Although the

four repeat expansion disease subcategories are based upon

presumed differences in their mechanisms of repeat mutation

toxicity, recent studies have revealed a number of shared

genomic features for repeat disease loci, irrespective of repeat

disease category, including (1) genetic instability characterized

by a strong tendency for repeats to further expand upon germ

line transmission (Pearson et al., 2005); (2) binding sites for the

multivalent transcription regulatory factor CTCF (Ohlsson et al.,

2001), within close proximity of the repeats (Filippova et al.,

2001); and (3) bidirectional transcription typically encompassing

the repeat itself (Batra et al., 2010). These features suggest that

certain epigenetic processes and chromatin regulatory path-

ways may be shared in common between different repeat

diseases. As the SCA7 CAG repeat is the most unstable of all

the CAG/polyQ repeat loci, and the SCA7 CAG repeat is closely

flanked by two functional CTCF binding sites, the SCA7 CAG

repeat is among the repeat disease loci likely to display this

constellation of genomic features.

In light of the importance of ataxin-7 normal function for

SCA7 disease pathogenesis and potentially for global transcrip-

tion regulation, we initiated a series of studies aimed at under-

standing how ataxin-7 gene expression is regulated. The

ataxin-7 CAG repeat tract and the start site of translation are

1072 Neuron 70, 1071–1084, June 23, 2011 ª2011 Elsevier Inc.

both located in exon 3, which is flanked by two functional

CTCF binding sites (Filippova et al., 2001). CTCF is a highly

conserved 11 zinc-finger protein that mediates a variety of tran-

scription regulatory functions, including transcription activation,

transcription repression, insulator-boundary domain formation,

and genomic imprinting (Phillips and Corces, 2009). When we

analyzed the ataxin-7 repeat region, we discovered evidence

for an alternative promoter just 50 to exon 3, and identified an

antisense non-coding RNA, SCAANT1 (for spinocerebellar

ataxia-7 antisense noncoding transcript 1) that is convergently

transcribed across exon 4, exon 3, and the alternative promoter.

To understand the role of CTCF in regulating ataxin-7 transcrip-

tion, we introduced ataxin-7 minigenes, containing the ataxin-7

repeat region with a CAG repeat expansion, into transgenic

mice. Studies of these transgenic mice and of human retinoblas-

toma cell lines revealed that CTCF binding is required for

production of SCAANT1, and that loss of SCAANT1 expression

de-repressed ataxin-7 sense transcription from the alternative

promoter. Although SCAANT1 expression in trans did not reduce

ataxin-7 alternative sense promoter activity in vitro or in vivo,

convergent transcription of SCAANT1 in cis led to repression

that was accompanied by posttranslational modification of

histones. Our studies reveal a regulatory pathway that links

CTCF transactivation of antisense noncoding RNA with repres-

sion of the corresponding sense transcript. The likely contribu-

tion of this pathway to SCA7 disease pathogenesis underscores

the potential importance of altered epigenetic regulation for

disease pathology at repeat loci characterized by bidirectional

transcription.

RESULTS

Identification of Overlapping Sense and AntisenseTranscripts at the Ataxin-7 LocusThe ataxin-7 CAG/polyglutamine (polyQ) repeat is located in the

first coding exon (i.e., exon 3) and is flanked by two CTCF

binding sites (Filippova et al., 2001). To determine the basis of

ataxin-7 gene expression regulation, we surveyed ataxin-7

human genomic sequence, including exon 3 and the CAG repeat

region, with the UCSC genome browser (http://genome.ucsc.

edu). Bioinformatics analysis of this region, using FirstEF

(Davuluri et al., 2001), revealed evidence for an alternative

promoter in the intron 50 to exon 3. The presence of a strong

peak for the promoter-associated H3K4me3 modification at

this location strongly supported the existence of this alternative

promoter (see Figure S1 available online). When we analyzed

mouse ataxin-7 genomic sequence, we found that the transcrip-

tion start site (TSS) for the murine ataxin-7 gene is located in the

ataxin-7 repeat region in close proximity to the alternative

promoter predicted for the human gene. Interestingly, the previ-

ously defined human ataxin-7 TSS, which is located >40 kb 50 tothis region, annotated on the UCSC genome browser, and vali-

dated by RLM-RACE (Figure S1), is not predicted as a promoter

or TSS in mouse (http://genome.ucsc.edu). To evaluate this

prediction, we performed RLM-RACE on murine RNA samples

and identified a cluster of TSSs located 85, 100, and 255 nucle-

otides 50 to the annotated mouse ataxin-7 TSS. However, unlike

in the human, there is no distant upstream ataxin-7 TSS in mice.

http://genome.ucsc.edu



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Figure 1. The Ataxin-7 Gene Contains an Alterna-

tive Sense Promoter

(A) Diagram of the genomic region of the human ataxin-7

gene and constructs generated to map the alternative

sense promoter. Boxes represent exons, while solid lines

correspond to introns. The alternative promoter is indi-

cated as P2A and is predicted to produce a transcript that

contains an alternatively spliced intron (gray box). Various

genomic fragments, containing 10 CAG/CTG repeats,

were cloned into a firefly luciferase vector to yield the

different luciferase expression constructs shown here.

(B) Transactivation assay results for human ataxin-7

genomic fragments. The luciferase expression constructs

shown in (A) were transfected into primary astrocytes,

along with Renilla luciferase constructs for normalization,

and relative luminescence unit (RLU) measurements were

obtained. EV corresponds to empty vector, all experi-

ments were performed in quadruplicate, and errors bars =

SEM. RLU activity obtained for the vector 1F construct

corresponded to �10% of a positive control CMV-eGFP-

IRES-luciferase vector (not shown). See Figure S1.

Neuron


Thus, the human ataxin-7 gene contains two promoters: the

standard, upstream ‘‘P1’’ promoter and the alternative ‘P2A’

promoter (Figure S2), while the mouse ataxin-7 gene contains

only one promoter—P2A.

Although computational algorithms for identification of

promoters and TSSs are powerful approaches for defining regu-

latory regions, experimental validation is necessary. To confirm

the promoter calls, and to define the location of regulatory

elements in this region, we cloned a series of human ataxin-7

CAG10 genomic fragments into a luciferase reporter (Figure 1A)

and transfected the different ataxin-7 genomic fragment—lucif-

erase constructs into primary cerebellar astrocytes. When we

measured relative luciferase activity, we detected robust lucif-

erase transactivation for ataxin-7 genomic fragments containing

sequences just 50 to the newly discovered TSS (Figure 1B). To

define the alternative promoter, which we labeled ‘‘P2A,’’ we

performed 50 RACE and found that the alternative ataxin-7 TSS

is located at the 30 end of intron 2. Sequencing of 50 RACE clones

revealed that transcripts initiating from P2A contain a single first

exon comprising the last �400 bp of intron 2 and exon 3, or

Neuron 70, 107

instead undergo splicing to join a shorter exon

(‘‘2A’’) with exon 3 (Figure 1A).

In the course of inventorying ESTs in the

ataxin-7 repeat region, we discovered an EST

(BU569004) in antisense orientation. Analysis

of the sequence in this region revealed that

the 50 end of this EST corresponded to a NotI

restriction site, and that the 30 end of this EST

colocalized with a putative polyadenylation

signal sequence in antisense orientation (Fig-

ure 2A). As NotI digests were typically used

during the former era of EST discovery, the 50

end of this transcript was likely created during

its cloning. To identify the extent of the anti-

sense transcript, we performed strand-specific

RT-PCR and 50 RACE, and mapped the TSS to

intron 4 (Figure 2A). We named this antisense

transcript SCAANT1 for ‘‘spinocerebellar ataxia-7 antisense

noncoding transcript 1.’’ To delineate the regulatory region

responsible for transcription of SCAANT1, we cloned a series

of human ataxin-7 CAG10 genomic fragments into a luciferase

reporter construct in antisense orientation (Figure 2A) and trans-

fected the different ataxin-7 antisense genomic fragment—lucif-

erase constructs into primary cerebellar astrocytes. We noted

that a short stretch of DNA 50 to the SCAANT1 TSS was required

for transactivation, while a sizable sequence 30 to the SCAANT1

TSS was needed to achieve robust transactivation (Figure 2B).

As the two CTCF binding sites lie within the regulatory domain

mapped by the luciferase reporter assays, we derived another

set of luciferase reporter constructs, based upon our most

potent construct 2R, in which we mutated either of the CTCF

binding sites (Figure 2A). When we measured the transactivation

competence of the 2R-m2 and 2R-m1 constructs, we observed

marked reductions in luciferase activity (Figure 2B), suggesting

that CTCF binding site integrity is required for maximal

SCAANT1 expression. We also derived an ataxin-7 antisense

construct carrying a CAG92 repeat expansion (2R-exp), and

1–1084, June 23, 2011 ª2011 Elsevier Inc. 1073

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Figure 2. Identification and Mapping of an Anti-

sense ncRNA at the Human Ataxin-7 Locus

(A) Diagram of the genomic region of the human ataxin-7

gene, showing the human ataxin-7 antisense ncRNA

(SCAANT1), and the constructs generated to map the

SCAANT1 promoter. Boxes represent exons, while solid

lines correspond to introns. The location of BU569004—

an antisense EST, a predicted polyadenylation signal

sequence, the transcriptional domain for SCAANT1, and

two CTCF binding sites are shown. Various genomic

fragments in antisense orientation, all containing 10 CAG/

CTG repeats except for 2R-exp, were cloned into a firefly

luciferase vector to produce the different expression

constructs shown here.

(B) Transactivation assay results for human ataxin-7

antisense genomic fragments. The luciferase expression

constructs shown in (A) were transfected into primary

astrocytes, along with Renilla luciferase constructs for

normalization, and relative luminescence unit (RLU)

measurements were obtained. Construct 2R-m2 contains

amutated CTCF-II binding site, construct 2R-m1 contains

a mutated CTCF-I binding site, and construct 2R-exp

contains a 92 CAG/CTG repeat. EV corresponds to empty

vector, experiments were performed in quadruplicate,

and errors bars = SEM.

See Figure S2.

Neuron


when we measured its transactivation competence, we docu-

mented a significant reduction in luciferase activity (Figure 2B).

An Ataxin-7 Genomic Fragment Yields a SCA7Phenotype in Transgenic Mice upon Mutationof the 30 CTCF Binding SiteThe existence of an �1.4 kb antisense noncoding transcript

overlapping a potentially strong sense promoter at the human

ataxin-7 locus suggested that their transcription regulationmight

be linked. As CTCF binding site integrity was required for

SCAANT1 transcription, we derived two ataxin-7 minigene

constructs that contain the sense P2A promoter and SCAANT1,

flanked by �5 kb of DNA 50 to this region and�8 kb of DNA 30 tothis region (Figure S2). Within this 13.5 kb human ataxin-7

genomic fragment reside two CTCF binding sites, known as

CTCF-I and CTCF-II. To understand the regulatory relationship

between SCAANT1 and ataxin-7 transcription from promoter

P2A, we introduced an 11 nucleotide substitution mutation at

the 30 CTCF-I binding site (Figure S2). The location of the muta-


tion was based upon DNA footprinting analysis,

and validation of abrogated CTCF binding was

achieved by electrophoretic mobility shift

assays, as we have shown (Libby et al.,

2008). In this way, we derived two distinct

ataxin-7 genomic fragment constructs with an

expanded CAG repeat tract: SCA7-CTCF-I-wt

and SCA7-CTCF-I-mut (Figure S2). To deter-

mine the role of CTCF binding in regulating

ataxin-7 repeat instability and transcription,

we used these constructs to produce lines of

transgenic mice. We have shown that mutation

of the CTCF-I binding site significantly dimin-

ishes CTCF occupancy in vivo in the SCA7-CTCF-I-mut mice

by ChIP analysis and found that mutation of the CTCF-I binding

site leads to increased repeat instability in the germline and

somatic tissues (Libby et al., 2008). Further studies of these

mice also revealed that SCA7-CTCF-I-mut mice become tremu-

lous, display weight loss, and develop an unsteady gait at

5–9 months of age (Movie S1). This phenotype, which is

observed in both SCA7-CTCF-I-mut transgenic lines ((1) and

(2)), progresses to become a prominent gait ataxia until the

mice die prematurely at 8–14 months of age, with the SCA7-

CTCF-I-mut-(2) line exhibiting a more rapidly progressive and

severe phenotype. In contrast, four independent lines of SCA7-

CTCF-I-wt mice did not exhibit any physical or neurological

abnormalities, and have a normal lifespan.

As SCA7-CTCF-I-mut transgenic mice develop a pronounced

ataxia, reminiscent of the gait difficulties seen in SCA7 patients

and in other lines of SCA7 transgenic mice (La Spada et al.,

2001; Yoo et al., 2003), we performed histopathology studies

and behavioral testing. SCA7 patients develop a cone-rod

Figure 3. SCA7-CTCF-I-mut Mice Develop

Retinal and Cerebellar Degeneration,

Produce polyQ-Expanded Protein and

Suffer Loss of Ataxin-7 Antisense ncRNA

Expression

(A) Confocal microscopy of retinal whole-mounts

from 11-month-old SCA7-CTCF-I-mut (mut) and

SCA7-CTCF-I-wt (wt) mice. Retinas immuno-

stained with antibodies against red/green cone

photoreceptors (red) and rod photoreceptors

(green) reveal dramatic loss of cone photorecep-

tors and more moderate, but nonetheless marked,

drop-out of rods in SCA7-CTCF-I-mut mice.

(B) Confocal microscopy of cerebellar sections

from 11-month-old SCA7-CTCF-I-mut (mut) and

SCA7-CTCF-I-wt (wt) mice. Sections immuno-

stained with antibodies against calbindin (green)

and ataxin-7 (magenta), and counterstained with

DAPI (blue) reveal marked Purkinje cell degener-

ation, loss, and protein aggregate formation

(arrows) in SCA7-CTCF-I-mut mice.

(C) Western blot analysis of cortex protein lysates

obtained from six week-old SCA7-CTCF-I-wt

mice, two independent lines of SCA7-CTCF-I-mut

mice, and a nontransgenic control (Ntg) immuno-

blotted with 1C2 antibody that detects polyQ-

expanded protein. Both lines of SCA7-CTCF-I-

mut mice display an �47 kDa band (arrow) that is absent from the Ntg and barely visible in the SCA7-CTCF-I-wt mouse. The asterisk indicates a nonspecific

background band that confirmed equivalent loading.

(D) Reciprocal expression of sense and antisense ataxin-7 RNAs in three month-old SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice. We performed strand-

specific RT-PCR, and quantified RNA expression relative to 18SRNA. Results for three sets of brain RNAs per line are shown, with ataxin-7 sense RNA expression

arbitrarily set to 1 for SCA7-CTCF-I-wt mice, and ataxin-7 antisense (SCAANT1) RNA expression arbitrarily set to 1 for SCA7-CTCF-I-mut mice. Comparable

results were obtained when we normalized to GAPDH, and absolute values indicated that ataxin-7 antisense transcript levels are �2.1-fold higher than ataxin-7

sense levels in SCA7-CTCF-I-wt mice (not shown). Assays were performed in triplicate, and error bars = SEM.

(E) In situ hybridization of SCAANT1 in threemonth-old SCA7 transgenic mice. Prominent signal in the Purkinje cell layer of SCA7-CTCF-I-wtmice (wt) is detected

with the ataxin-7 antisense riboprobe; however, minimal labeling in the Purkinje cell layer of SCA7-CTCF-I-mut mice (mut) is observed with this riboprobe.

See Figure S3.

Neuron


dystrophy retinal degeneration, characterized by dramatic loss

of cone photoreceptors and visual dysfunction (Ahn et al.,

2005; To et al., 1993). To determine if SCA7-CTCF-I-mut mice

recapitulate this phenotype, we immunostained retinal whole-

mounts from age-matched SCA7-CTCF-I-mut and SCA7-

CTCF-I-wt mice, and observed a marked drop-out of cone

photoreceptors in SCA7-CTCF-I-mut mice (Figure 3A). Electro-

retinogram testing corroborated this finding, as SCA7-CTCF-I-

mut mice went blind with a degradation of cone responses

ahead of rod responses (Figure S3). The visible ataxia phenotype

in affected SCA7-CTCF-I-mut mice led us to compare cerebellar

sections from age-matched SCA7-CTCF-I-mut mice and SCA7-

CTCF-I-wt mice. This analysis revealed dramatic Purkinje cell

degeneration, as well as ataxin-7 positive aggregates in Purkinje

cells in SCA7-CTCF-I-mut mice (Figure 3B). These findings

confirm that mutation of the 30 CTCF binding site, within a human

ataxin-7 minigene lacking the canonical ataxin-7 TSS at exon 1,

is sufficient to recapitulate the SCA7 phenotype in independent

lines of transgenic mice.

Recapitulation of the SCA7 phenotype in SCA7-CTCF-I-mut

mice, together with the observation of ataxin-7-positive inclu-

sions in cerebellar Purkinje cells, suggested that mutation of

the 30 CTCF binding site had resulted in the initiation of sense

transcription within the ataxin-7 minigene construct. To test

this hypothesis, we performed RT-PCR analysis on SCA7-

CTCF-I-mut mice and detected expression of the ataxin-7 first

coding exon in RNA samples from cerebellum and cortex (data

not shown). As SCA7 disease pathogenesis typically involves

production of a misfolded polyQ-expanded ataxin-7 protein,

we performed western blot analysis with the 1C2 antibody that

is specific for expanded polyQ tracts (Trottier et al., 1995) and

observed an �47 kDa protein in brain lysates from each SCA7-

CTCF-I-mut transgenic line (Figure 3C). We noted higher expres-

sion in the SCA7-CTCF-I-mut-(2) line, consistent with its more

severe phenotype. Low-level expression of the �47 kDa protein

was detected in SCA7-CTCF-I-wt mice (Figure 3C), and this

�47 kDa protein product corresponds to an open reading frame

starting at the initiator ATG codon in exon 3 and continuing

through exon 4 until the first nonsense codon in intron 4. The

production of a protein product and disease phenotype in the

SCA7-CTCF-I-mut mice is reminiscent of the R6/2 mouse model

of HD, in which a small fragment from the htt gene was intro-

duced into mice to model repeat instability, but also yielded

a truncated protein product resulting in a HD-like phenotype—

despite the fact that the construct lacked a 30 polyadenylationsite or characterized promoter (Mangiarini et al., 1996).


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Figure 4. Reciprocal Ataxin-7 Sense and Antisense

Expression in Human Tissues and SCA7

(A) Results of qRT-PCR analysis of ataxin-7 sense tran-

script. Expression of ataxin-7 sense transcript was

significantly less in lung and kidney (p < 0.01 and p < 0.001

by ANOVA with post hoc Tukey test). Assays were per-

formed in triplicate, and error bars = SEM.

(B) Results of qRT-PCR analysis of ataxin-7 antisense

ncRNA SCAANT1. Expression of SCAANT1 was signifi-

cantly elevated in lung and kidney (p < 0.001 and p < 0.01

by ANOVA with post hoc Tukey test). Assays were per-

formed in triplicate, and error bars = SEM.

(C) RT-PCR analysis of SCA7 patient fibroblast RNAs.

RT-PCR amplification reveals the ataxin-7 antisense

ncRNA SCAANT1 from the normal allele in a control

fibroblast line (15Q) and SCA7 patient fibroblast line (55Q),

but not from the expanded allele for the SCA7 patient. RT-

PCR amplification of the ataxin-7 sense transcript,

however, indicates strong expression from the normal and

expanded allele in SCA7 fibroblasts.

(D) Quantification of ataxin-7 RNA levels in SCA7 patient

fibroblasts. Significant increases in ataxin-7 sense

expression were present in SCA7 patients carrying either

55Q or 150Q alleles (p < 0.01 by ANOVA). Experiments

were performed in triplicate, and error bars = SEM.

(E) Quantification of ataxin-7 RNA levels in SCA7 patient

lymphocytes. Significant increases in ataxin-7 sense

expression were present in SCA7 patients carrying 51Q,

59Q, or 66Q alleles (p < 0.01 by ANOVA). Experiments

were performed in triplicate, and error bars = SEM.

See Figure S4.

Neuron


Loss of SCAANT1 Expression Is Accompanied byDerepression of Sense TranscriptionOur findings indicate that mutation of the 30 CTCF binding site is

responsible for initiation of robust sense transcription in SCA7-

CTCF-mut-I mice, as SCA7-CTCF-I-wt mice carrying an

ataxin-7 genomic fragment with an intact 30 CTCF binding site

express low levels of ataxin-7 mRNA and protein. To determine

if the levels of ataxin-7 sense and antisense transcription within

the repeat region domain correlate in SCA7-CTCF-I-wt and

SCA7-CTCF-I-mut mice, we performed quantitative strand-

specific RT-PCR amplification, and detected ataxin-7 sense

and antisense transcripts in each line. We found that ataxin-7

sense transcript levels were elevated �370-fold in the brains of

SCA7-CTCF-I-mut mice compared to SCA7-CTCF-I-wt mice,

and this was accompanied by an �140-fold decrease in

SCAANT1 expression (Figure 3D). In situ hybridization analysis

confirmed robust expression of SCAANT1 in the cerebellum of

SCA7-CTCF-I-wt mice but did not detect strong SCAANT1

expression in SCA7-CTCF-I-mut mice (Figure 3E). In situ hybrid-


ization analysis indicated moderate to strong

expression of SCAANT1 in SCA7-CTCF-I-wt

mice throughout the brain (Figure S4A). Corre-

spondingly, in situ hybridization did not yield

evidence for much SCAANT1 expression in

the brain of SCA7-CTCF-I-mut mice (Fig-

ure S4B). Taken together, these findings show

that reduced SCAANT1 expression correlates

with increased P2A promoter activity, resulting in increased

sense expression of the ataxin-7 gene.

Ataxin-7 Sense Expression and SCAANT1 ExpressionAre Inversely Correlated in Human Tissues and SCA7PatientsOur studies of the SCA7-CTCF-I-wt and SCA7-CTCF-I-mutmice

suggested that expression of the ataxin-7 sense transcript

inversely correlates with expression of SCAANT1. To determine

if this reciprocal expression relationship exists in normal human

tissues, we performed qRT-PCR analysis on a panel of human

tissue RNAs.While we documented high levels of ataxin-7 sense

transcript in cortex, cerebellum, striatum, and liver, we found

much lower levels of ataxin-7 in lung and kidney (Figure 4A).

Interrogation of SCAANT1 expression levels revealed an oppo-

site pattern, as SCAANT1 was much higher in the lung and

kidney than in cortex, cerebellum, striatum, or liver (Figure 4B).

As the presence of the CAG repeat expansion decreased

SCAANT1 promoter activity in our luciferase reporter assays

Neuron


(Figure 2), we tested if the diametrically opposed expression of

ataxin-7 sense transcript and SCAANT1 might occur in the

context of SCA7 disease. To test this hypothesis, we performed

RT-PCR analysis of a SCA7 patient fibroblast cell line, and while

we could amplify both the normal and expanded repeat alleles

for the ataxin-7 sense transcript, we could not detect antisense

SCAANT1 transcript expression from the expanded 55Q allele

(Figure 4C). We then performed quantitative RT-PCR analysis

of ataxin-7 sense expression on fibroblasts obtained from two

SCA7 patients, one with a moderately sized disease repeat

(55Q), and one with a severely expanded repeat (150Q). With

increasing expansion size, we observed significantly increased

ataxin-7 sense transcript levels (Figure 4D), indicating that

expansion of the CAG repeat at the ataxin-7 locus yields

increased levels of ataxin-7 transcript in association with

reduced expression of SCAANT1. We also obtained a set of

peripheral blood samples from three additional SCA7 patients,

isolated RNA from their lymphocytes, and performed RT-PCR

analysis. Antisense SCAANT1 transcript expression could not

be detected from the expanded allele of the SCA7 samples,

and all three SCA7 patients exhibited significantly increased

ataxin-7 sense transcript levels (Figure 4E).

CTCF Regulates SCAANT1 Expression and Ataxin-7Alternative Sense TranscriptionAs mutation of the 30 CTCF binding site reduced the activity of

the SCAANT1 promoter while derepressing ataxin-7 sense

expression from promoter P2A, we hypothesized that CTCF

modulates ataxin-7 sense expression from this promoter by

driving the expression of SCAANT1. To test this hypothesis,

we validated two different CTCF shRNAs and derived a dual

CTCF knockdown vector. After subcloning the CTCF dual

shRNA knockdown fragment into a lentiviral construct with

a linked eGFP expression cassette, we infected human Y-79 reti-

noblastoma cells and isolated RNA from flow-sorted GFP-posi-

tive Y-79 cells. Real-time RT-PCR analysis confirmed CTCF

knockdown, and revealed a significant reduction in the expres-

sion level of SCAANT1 (Figure 5A). Significant reduction in

SCAANT1 expression was accompanied by a marked increase

in the ataxin-7 sense transcript from the P2A promoter, but not

from the previously defined ‘‘standard’’ ataxin-7 sense promoter,

located >40 kb 50 to the repeat region (Figure 5A). Although

direct, physiological comparison of the standard P1 promoter

and P2A promoter is complicated by the coexistence of

SCAANT1 transcription, analysis of ataxin-7 alternative sense

and standard sense expression at baseline in Y-79 cells revealed

only modestly (i.e., �2.5-fold) higher levels of the ataxin-7 stan-

dard sense transcript, consistent with the similar degree of

H3K4me3enrichment noted for their respective TSSs (FigureS1).

To examine the trans contribution of CTCF protein levels to the

regulation of ataxin-7 gene expression in the SCA7-CTCF-I-

mut mice, we generated CTCF heterozygous knock-out mice

(G.N.F. et al., unpublished data). ChIP analysis has indicated

that reduced CTCF occupancy at the mutated 30 CTCF binding

site occurs in the SCA7-CTCF-I-mut mice (Libby et al., 2008),

and this may account for the de-repression of ataxin-7 sense

expression from promoter P2A. To test if the effect of this cis

mutation could be compounded by reduction of CTCF expres-

sion in trans, we crossed SCA7-CTCF-I-mut mice with CTCF

heterozygous null mice, and compared the resulting SCA7-

CTCF-I-mut; CTCF+/� mice with their SCA7-CTCF-I-mut;

CTCF+/+ littermates. We confirmed reduced dosage of CTCF ex-

pression in the SCA7-CTCF-I-mut; CTCF+/� mice, and observed

significantly reduced expression of the antisense SCAANT1 tran-

script (Figure 5B). This was accompanied by increased ataxin-7

sense expression (Figure 5B), which yielded a more rapidly

progressive retinal phenotype in SCA7-CTCF-I-mut; CTCF+/�

mice (Figures 5C–5E). The worsened phenotype was also

reflected by a significantly shortened life span (Figure 5F). Hence,

decreased expression of CTCF agonized the SCA7 phenotype in

SCA7-CTCF-I-mut mice by further de-repressing ataxin-7 P2A

promoter activity. As cohesin may play a role in CTCF insulator

formation (Parelho et al., 2008; Stedman et al., 2008; Wendt

et al., 2008), we performed ChIP analysis for two cohesin

subunits in the SCA7 transgenic mice, and observed reduced

occupancy of SMC1 and SMC3 at the 30 CTCF binding site in

the cerebellum of SCA7-CTCF-I-mut mice (Figure S5), suggest-

ing that cohesin may also participate in CTCF-mediated tran-

scription regulation at the ataxin-7 locus.

SCAANT1 Mediates Repression of the CorrespondingAtaxin-7 Sense Transcript in cis

CTCF binding regulates sense and antisense transcription at the

ataxin-7 locus, and expression levels of the ataxin-7 sense tran-

script and antisense SCAANT1 message are inversely corre-

lated. Hence, a key question is whether SCAANT1 transcription

is coincident, or required for derepression of ataxin-7 sense

promoter P2A. To determine if SCAANT1 transcription is

necessary for the regulation of ataxin-7 sense expression, and

to distinguish between a cis or trans regulatory mechanism, we

developed a CMV-SCAANT1 expression construct. We then

cotransfected astrocytes with a highly active ataxin-7 genomic

fragment—luciferase reporter construct and the CMV-SCAANT1

expression construct and tested if enforced expression of

SCAANT1 would downregulate ataxin-7 sense P2A promoter

activity, but we observed no effect (Figure 6A). This in vitro

experiment was paralleled by an in vivo study in which we

crossed SCA7-CTCF-I-mut mice with SCA7-CTCF-I-wt mice,

reasoning that the dramatically elevated levels of SCAANT1 anti-

sense RNA in SCA7-CTCF-I-wt mice would reduce ataxin-7

sense expression in SCA7-CTCF-I-mut mice, if SCAANT1

regulation of ataxin-7 sense expression is occurring in trans.

However, behavioral analysis revealed further worsening in

rotarod performance in SCA7-CTCF-mut-I; SCA7-CTCF-I-wt

mice in comparison to SCA7-CTCF-I-mut mice (Figure S6).

Furthermore, SCA7-CTCF-I-mut; SCA7-CTCF-I-wt bigenic

mice displayedmore severe Purkinje cell degeneration than their

singly transgenic SCA7-CTCF-I-mut littermates (Figures 6B–6D).

Thus, greater expression of SCAANT1 in SCA7-CTCF-I-mut;

SCA7-CTCF-I-wt mice did not reduce ataxin-7 sense transcrip-

tion; instead, low levels of ataxin-7 sense protein product from

the SCA7-CTCF-I-wt mice (Figure 3C) enhanced the SCA7

phenotype in bigenic SCA7-CTCF-I-mut; SCA7-CTCF-I-wt

mice.

To test if SCAANT1 transcription regulates promoter P2A

in cis, we engineered ataxin-7 P2A-exon 3(CAG10)-exon 4


A

B

F

Weeks

Su

rviv

al ra

te

SCA7-CTCF-I-mut;CTCF +/-

SCA7-CTCF-I-mut;CTCF +/+

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

CTCF Antisense Alt. sense Standard sense

ControlLenti-CTCF-shRNA

Exp

ress

ion

leve

l (r

elat

ive

to 1

8S R

NA

)

****

*

SCA7-CTCF-I-mut; CTCF +/-

SCA7-CTCF-I-mut; CTCF +/+

CTCF Antisense

Exp

ress

ion

leve

l (r

elat

ive

to A

CT

B)

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Exp

ression

level (relative to

GA

PD

H)

* *

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

*

Sense

Figure 5. CTCF Regulates Promoter Usage

for Ataxin-7 Sense and Antisense Tran-

scripts

(A) CTCF knockdown in Y-79 retinoblastoma cells

leads to decreased ataxin-7 antisense RNA

expression and increased exon 2A transcript

expression. Human Y-79 retinoblastoma cells

were infected with a lentiviral expression con-

struct containing two tandem shRNAs targeting

CTCF (lenti-CTCF-shRNA) or with empty vector

(Control), and expression levels were measured by

qPCR. Marked reductions in expression of CTCF

and ataxin-7 antisense SCAANT1 transcripts were

noted (p < 0.01 by ANOVA) andwere accompanied

by a significant increase in the ataxin-7 alternative

sense transcript (p < 0.05 by ANOVA). Knockdown

of CTCF did not affect expression of the ataxin-7

transcript initiated from a human-specific ataxin-7

promoter located >40 kb 50 to the alternative

promoter. Experiments were performed in tripli-

cate, the expression level for each transcript upon

CTCF knockdown is normalized to its respective

control, and error bars = SEM.

(B) Decreased CTCF dosage reduces SCAANT1

expression in SCA7-CTCF-I-mut mice. We

crossed SCA7-CTCF-I-mut mice with CTCF+/�

mice and derived SCA7-CTCF-I-mut mice on

a CTCF heterozygous null background (CTCF+/�)or a wild-type background (CTCF+/+). We per-

formed qRT-PCR analysis of CTCF in the brain of

three-month-old mice and confirmed reduced

expression of CTCF in SCA7-CTCF-I-mut;

CTCF+/� mice (p < .0.05 by ANOVA). When we

measured brain expression of SCAANT1 (Anti-

sense) transcript and ataxin-7 sense transcript by

qRT-PCR, we noted a marked reduction in anti-

sense expression and a significant increase in

sense expression in SCA7-CTCF-I-mut; CTCF+/�

mice (p < .0.05 by ANOVA). Experiments were

performed in triplicate, the expression level for

each transcript is normalized to the SCA7-CTCF-I-

mut; CTCF+/+ level, and error bars = SEM.

(C–E) Reduced CTCF dosage enhances SCA7

retinal degeneration in SCA7-CTCF-I-mut mice.

Confocal microscopy of retinal sections from six-

month-old nontransgenic controls (C), SCA7-

CTCF-I-mut; CTCF+/� (D), and SCA7-CTCF-I-mut;

CTCF+/+ (E), reveals more severe cone photore-

ceptor dropout in SCA7-CTCF-I-mut mice on

a CTCF heterozygous null background. Red/green pigment antibody = red; propidium iodide = cyan. OS = outer segments; ONL = outer nuclear layer; INL = inner

nuclear layer; GCL = ganglion cell layer. Scale bar corresponds to 50 mM.

(F) Kaplan-Meier survival analysis of SCA7-CTCF-I-mut; CTCF+/�mice.We recorded lifespan for cohorts ofSCA7-CTCF-I-mut; CTCF+/�mice (n = 28) andSCA7-

CTCF-I-mut; CTCF+/+ mice (n = 40) and observed a significant reduction in life span for SCA7-CTCF-I-mut mice on a CTCF heterozygous null background

(p < 0.05 by log-rank test).

See Figure S5.

Neuron


genomic fragment constructs with a 30 IRES-luciferase (luc) in

sense orientation, and a Renilla luciferase (Rluc) in antisense

orientation (Figure 6E). We replaced the antisense SCAANT1

promoter with a tet-regulatable element (TRE) to yield the

‘‘TRE-only’’ ataxin-7 genomic fragment construct, and then

created a second version by cloning a polyA transcription termi-

nation signal (‘‘polyA trap’’) in antisense orientation into exon 3

(Figure 6E). To confirm the integrity of the polyA trap, we trans-

fected astrocytes with either the TRE-only or TRE-polyA-trap


ataxin-7 vector, induced with doxycycline and observed marked

reduction of Rluc/luc for the TRE-polyA-trap ataxin-7 vector

(Figure 6F). When we measured ataxin-7 expression by qRT-

PCR, we observed significant derepression of ataxin-7 sense

expression in TRE-polyA-trap ataxin-7 transfected cells (Fig-

ure 6G). Hence, premature termination of SCAANT1 transcrip-

tion released repression of ataxin-7 P2A promoter activity,

indicating that SCAANT1 regulates ataxin-7 sense expression

in cis by convergent transcription.

Figure 6. The Ataxin-7 Antisense ncRNA,

SCAANT1, Regulates Ataxin-7 Alternative

Promoter Activity in cis, but Not in trans

(A) Effect of enforced expression of SCAANT1

upon transactivation competence of ataxin-7

promoter P2A. The human ataxin-7 genomic

fragment luciferase expression construct (1F; see

Figure 1A) was transfected into primary astrocytes,

along with Renilla luciferase, and a CMV expres-

sion construct containing SCAANT1 (CMV-

SCAANT1). Relative luminescence unit (RLU)

measurements were obtained. EV corresponds to

the empty luciferase vector, CMV-empty refers to

the empty CMV expression construct, all experi-

ments were performed in quadruplicate, and errors

bars = SEM.

(B–D) Effect of overexpression of SCAANT1 upon

the de-repressed ataxin-7 alternative promoter

in vivo. We crossed the SCA7-CTCF-I-mut mice

with SCA7-CTCF-I-wt mice, and compared cere-

bellar histopathology between littermates of the

following genotypes: SCA7-CTCF-I-wt (B), SCA7-

CTCF-I-mut (C), and SCA7-CTCF-I-mut; SCA7-

CTCF-I-wt (D). Here, we see the results of confocal

microscopy of cerebellar sections immunostained

with antibodies against calbindin (green) and

ataxin-7 (magenta), and counterstained with DAPI

(blue) for mice at 7 months of age. Strong calbindin

immunoreactivity is observed in SCA7-CTCF-I-wt

mice (B), while an obvious reduction in calbindin

immunoreactivity is apparent in the SCA7-CTCF-I-

mut mice (C). Calbindin immunoreactivity is further

reduced in SCA7-CTCF-I-mut; SCA7-CTCF-I-wt

bigenic mice, and this is accompanied by an

increased accumulation of aggregated ataxin-7

protein (magenta puncta) (D), indicating that

enforced in vivo expression of SCAANT1 does

not promote degradation of the complementary

ataxin-7 sense transcript. Rather, crossing with

SCA7-CTCF-I-wt mice actually worsens the

phenotype, since SCA7-CTCF-I-wt mice produce

modest levels of polyQ-expanded ataxin-7 protein

(see Figure 3C).

(E) Diagram of the ataxin-7 antisense premature

termination construct. We replaced the ataxin-7

antisense ncRNA promoter with a tet-responsive

element—minimal CMV promoter (TRE) and in-

serted Renilla luciferase (R-luc) in antisense orientation to quantify the activity of the TRE promoter. The IRES-luc segment is cloned in sense orientation to permit

normalization. The poly-A transcription termination cassette was cloned into exon 3 in antisense orientation (asterisk), and we prepared two versions of this

construct: either without the poly-A cassette (TRE-only) or with the poly-A cassette (TRE-poly-A-trap).

(F) Validation of poly-A transcription termination activity. The TRE-only construct or the TRE-poly-A-trap construct was transfected into primary astrocytes, along

with a CMV-tet-activator expression construct, and Renilla luciferase (R-luc) and luciferase (luc) activities were measured. A marked reduction in the R-luc / luc

ratio in the TRE-poly-A-trap construct was noted (p < 0.01, ANOVA).

(G) Convergent transcription is required for repression of ataxin-7 alternative promoter by the antisense ncRNA. The TRE-only construct or the TRE-poly-A-trap

construct was transfected into primary astrocytes, along with tet-activator, and ataxin-7 sense transcript levels were measured by qPCR. Ataxin-7 sense

expression was dramatically increased in cells transfected with the TRE-poly-A-trap construct (p < 0.01, ANOVA).

See Figure S6.

Neuron


Ataxin-7 Antisense Expression Yields RepressiveChromatin Modifications in Transgenic MiceSilencing of ataxin-7 sense P2A promoter activity by convergent

transcription of the SCAANT1 RNA raised a number of questions

as to the mechanism of repression. We hypothesized that one

possibility might be chromatin-dependent gene silencing and

proceeded to evaluate the status of covalent histone modifica-

tions known to correlate with transcription activation and repres-

sion in SCA7 CTCF-I-mut and SCA7-CTCF-I-wt mice. To do this,

we performed ChIP analysis of histone marks, and interrogated

covalent histone modification status at amplicons spanning the

ataxin-7 repeat region, including the transcription domains and

start sites for the sense P2A promoter and SCAANT1 (Figure 7A).

Quantitative PCR analysis revealed highly enriched levels of the


A

B C DA E

ATG

(CAG)n


P2A

50 bp

> 38 kb to exon 5

>13 kb to exon 2 AAGTAAA

CTCF-II CTCF-I

AAAAA

6

B

0

4

1

Amplicon ‘A’ Amplicon ‘B’ Amplicon ‘C’

2

3

5

Amplicon ‘D’ Amplicon ‘E’

IP /

inp

ut:

H3K

27m

e3

(no

rmal

ized

to

c-M

yc C

TC

F s

ite)

***

**

**

0

4

1

Amplicon ‘A’ Amplicon ‘B’ Amplicon ‘C’

2

3

5

Amplicon ‘D’ Amplicon ‘E’

IP /

inp

ut:

H3K

9/14

ac

(no

rmal

ized

to

c-M

yc C

TC

F s

ite) 6

*

*C

SCA7-CTCF-I-wt

SCA7-CTCF-I-mut

SCA7-CTCF-I-wt

SCA7-CTCF-I-mut

Figure 7. ChIP Analysis of Histone Modification Status at the

Ataxin-7 Alternative Promoter Region in SCA7 Transgenic Mice

(A) Diagram of the genomic region of the human ataxin-7 gene, where the

alternative promoter (P2A), first coding exon, CTCF binding sites, and anti-

sense ncRNA, SCAANT1, are located. Boxes represent exons, while solid lines

correspond to introns. The positions of the five amplicons employed in the

ChIP analysis are indicated.

(B and C) Results of ChIP analysis of histone marks in the cerebellum of SCA7

transgenic mice. ChIP was performed with antibodies against a repressive

mark (H3K27me3) and activated transcription mark (H3K9/14Ac) on cerebellar

DNAs isolated from six-month old SCA7-CTCF-I-wt and SCA7-CTCF-I-mut

mice. (B) In SCA7-CTCF-I-wt mice that express minimal amounts of ataxin-7

sense RNA from P2A, there was significant enrichment of H3K27me3 ex-

tending from the alternative promoter region into intron 3 of the ataxin-7 gene

(p < 0.01 to 0.001, ANOVA). (C) This pattern was reversed in SCA7-CTCF-I-mut

mice that display robust activation of the P2A promoter, as significant

enrichment of the H3K9/14Ac mark was present in the ataxin-7 sense P2A

region and initial transcript region (p < 0.05, ANOVA). Results represent three

independent experiments quantifying histone antibody ChIP normalized to the

c-Myc promoter region. Error bars = SEM.

Neuron


repressive H3K27me3 mark at amplicons 50 and 30 to the P2A

TSS and also showed low levels of the active H3K9/14ac mark

in SCA7-CTCF-I-wt mice (Figures 7B and 7C). However, ChIP

analysis revealed a reversal of this pattern in the SCA7-CTCF-

I-mut mice, as amplicons adjacent to promoter P2A exhibited

very low levels of H3K27me3 associated with elevated levels of

H3K9/14ac (Figures 7B and 7C), consistent with active transcrip-

tion at promoter P2A. Hence, our survey of covalent histone

modifications in the ataxin-7 mini-gene mice supported a role

for chromatin-dependent gene silencing by SCAANT1.


DISCUSSION

Bidirectional transcription at repeat loci is emerging as an impor-

tant theme in repeat expansion diseases, including myotonic

dystrophy 1 (DM1), spinocerebellar ataxia type 8 (SCA8), the

fragile X syndrome of mental retardation, Friedreich’s ataxia

(FRDA), Huntington’s disease (HD), and Huntington’s disease-

like 2 (HDL2) (Mirkin, 2007). At the same time, a role for CTCF

in regulating chromatin structure and transcription at such repeat

disease loci is being recognized (La Spada and Taylor, 2010). At

the SCA7 locus, the significance of CTCF for regulating repeat

instability was recently demonstrated, and shown to involve

epigenetic processes (Libby et al., 2008). In this study, we

examined the ataxin-7 repeat region where the CTCF binding

sites reside, and discovered that ataxin-7 gene expression is

governed by an antisense ncRNA transcript. This transcript,

which we named ‘‘SCAANT1,’’ appears to regulate a previously

unrecognized ataxin-7 sense promoter by convergent transcrip-

tion that overlaps the ataxin-7 repeat and the adjacent P2A

sense promoter. Our studies thus reveal a pathway for regulating

ataxin-7 gene expression at this promoter via an antisense RNA

and link CTCF transactivation of SCAANT1with repression of the

convergently transcribed sense domain (Figure 8).

Repeat tracts can greatly influence chromatin structure, espe-

cially if they are CG-rich (Wang et al., 1996). The mapping of

CTCF binding sites in close proximity to such repeats suggested

the need to insulate surrounding DNA from the potentially

untoward effects of repeat-induced changes upon chromatin

structure. CTCF is a multivalent transcription regulatory factor,

known to possess enhancer-blocking activity (Phillips and

Corces, 2009). CTCF may also prevent inactivation of gene

expression, as CTCF can restrict the spread of X-inactivation,

thereby preserving the transcriptional activity of ‘‘escape’’ genes

(Filippova et al., 2005). Previous studies of repeat disease loci

have shown that CTCF can prevent epigenetic changes associ-

ated with heterochromatin formation and gene inactivation by

constraining antisense transcription (Cho et al., 2005; De Biase

et al., 2009; Filippova et al., 2005). We evaluated the role of

CTCF in regulating ataxin-7 gene expression from an adjacent

alternative promoter (P2A) by introducing two different ataxin-

7 minigenes into mice. These minigenes are �13.5 kb ataxin-7

genomic fragments that contain the P2A promoter, the

SCAANT1 domain, the ataxin-7 start site of translation, and

the CAG repeat tract. The two minigenes were identical except

for the presence of a substitution mutation in the ‘‘SCA7-

CTCF-I-mut’’ construct at the 30 CTCF binding site. Importantly,

this substitution mutation had been shown to completely abro-

gate CTCF binding (Libby et al., 2008). The SCA7-CTCF-I-wt

construct yielded four independent lines of transgenic mice

that did not develop a phenotype, despite possessing a

CAG92 repeat tract in the fully intact ataxin-7 minigene. Instead,

two independent lines of SCA7-CTCF-I-mut mice developed

a SCA7-like phenotype, characterized by cone-rod dystrophy

retinal degeneration and cerebellar atrophy. Further studies

indicated that loss of CTCF binding results in dramatically

reduced expression of SCAANT1 in association with high-level

ataxin-7 expression from the newly discovered alternative sense

promoter. Our findings thus reveal that CTCF does regulate

(CAG)nSense

Promoter

AntisensePromoter

Ataxin-7

CTCF CTCF

(CAG)nSense

Promoter

AntisensePromoter

Ataxin-7

(CAG)nSense

Promoter

AntisensePromoter

Ataxin-7

CTCF

(CAG)nSense

Promoter

Ataxin-7

AntisensePromoter

(CAG)nSense

Promoter

Ataxin-7 AntisensePromoter

Atx7Atx7 Atx7Atx7

Atx7Atx7Atx7Atx7

Atx7Atx7

Atx7Atx7

Atx7Atx7Atx7Atx7

A B

CTCF CTCF

(CAG)nSense

Promoter

AntisensePromoter

Ataxin-7

CTCF CTCF

Atx7Atx7

Atx7Atx7

Figure 8. Model for Transcription Regula-

tion at the Ataxin-7 Locus

(A) CTCF-induced, SCAANT1-mediated, Dicer-

dependent repression of ataxin-7 sense tran-

scription. The ataxin-7 CAG repeat is flanked by

CTCF binding sites. When the CTCF binding sites

are unoccupied, the adjacent ataxin-7 promoter is

active, and covalent histone modification at the

promoter region is consistent with active tran-

scription (light green dots). When CTCF levels

increase and CTCF binds, the antisense promoter

for the noncoding RNA SCAANT1 is activated,

yielding convergent transcription. This is accom-

panied by a transition from an active chromatin

conformation to a repressed chromatin state (red

dots), silencing ataxin-7 expression from sense

promoter P2A.

(B) CAG repeat expansion or CTCF reduction can

de-repress ataxin-7 sense expression. Under

conditions of modest CTCF expression, the

ataxin-7 sense promoter is moderately active (light

green dots). However, if the CAG repeat is

expanded, or CTCF levels dwindle, the flanking

CTCF binding sites become unoccupied, resulting

in reduced activity of the SCAANT1 promoter.

Under these conditions, the sense promoter is fully

derepressed, with a chromatin conformation

consistent with highly active transcription (green

dots), yielding robust expression of ataxin-7.

Neuron


ataxin-7 gene expression; however, instead of preventing tran-

scription repression, CTCF supports it. Furthermore, rather

than restricting antisense expression, CTCF promotes it.

Surveys of mammalian transcriptomes are uncovering

tremendous numbers and varieties of noncoding RNAs, and

the production of antisense transcripts appears to be a pervasive

feature of the human and mouse transcriptomes (He et al., 2008;

Kapranov et al., 2007; Okazaki et al., 2002). Whenwe discovered

that SCAANT1 expression levels inversely correlate with ataxin-7

sense expression in both SCA7 transgenic mice and human

tissues, we considered the possibility that SCAANT1 might be

regulating the expression of its sense counterpart, as reciprocal

expression of sense and antisense transcripts has been reported

for a number of human andmouse genes (Katayama et al., 2005).

Indeed, at the human p15 locus, gene silencing of sense expres-

sion by an antisense RNA has been documented and can be

achieved by enforced expression of the antisense transcript

(Yu et al., 2008). We tested if SCAANT1 expression in trans can

downregulate ataxin-7 alternative sense promoter activity in

luciferase reporter assay experiments and by crossing SCA7-

CTCF-I-mut mice with SCA7-CTCF-I-wt mice, as the latter

exhibit high-level SCAANT1 expression. However, SCAANT1

transcript elevation had no effect upon ataxin-7 alternative sense

expression in vitro or in vivo. Studies of antisense transcripts in

mice and humans, as well as other eukaryotes such as yeast,

have revealed evidence for inhibition of transcription by virtue

of actual transcription interference, when RNA polymerases

moving in opposite directions collide with one another (Osato

et al., 2007; Shearwin et al., 2005). To test if SCAANT1 regulates

sense expression in cis, we engineered an ataxin-7 genomic

fragment construct with a transcription terminator positioned in

the antisense orientation, and placed antisense transcription

under the control of an inducible promoter. After validating the

efficiency of the transcription terminator, wemeasured the effect

of premature transcription termination upon SCAANT1’s ability

to repress ataxin-7 sense expression, and we noted a dramatic

derepression of sense transcription, when antisense transcrip-

tion was prematurely terminated. These results confirmed that

SCAANT1 is directly regulating ataxin-7 alternative sense

expression and demonstrated that the regulation occurs in cis.

One obvious question, based upon our results is: Why does

the cell need such a complicated pathway for adjusting ataxin-

7 expression? Ataxin-7 is a core, and likely essential, component

of different ubiquitously expressed transcription coactivator

complexes (Helmlinger et al., 2004; Palhan et al., 2005; Zhao

et al., 2008). As deletion of the ataxin-7 ortholog Sgf73 eliminates

Ubp8-mediated histone deubiquitination in yeast (Kohler et al.,

2008), and knockdown of ataxin-7 results in disassembly of the

STAGA complex in mammals (Palhan et al., 2005), tight regula-

tion of ataxin-7 expression could be a mechanism for controlling

the activity of these coactivators. CTCF is a master regulator of

transcription, and its expression cannot be significantly adjusted

without killing the cell. However, minor changes in CTCF levels,

or binding activity, could have a dramatic impact upon the tran-

scriptional activity of the cell through its regulation of ataxin-7

expression, since ataxin-7 expression alterations would be

amplified by affecting the stability and function of entire co-acti-

vator complexes. Thus, CTCF control of ataxin-7 levels could

serve as a rheostat for setting global transcription activity status

for the cell.

Another important implication of our work is its relevance to

SCA7 disease pathogenesis and repeat disease biology. As we


Neuron


have shown, expansion of the ataxin-7 CAG repeat tract reduces

SCAANT1 promoter activity, resulting in minimally detectable

levels of SCAANT1 from the expanded allele in SCA7 patient

fibroblasts. This reduction in SCAANT1 expression derepresses

the ataxin-7 alternative promoter, and significantly boosts the

level of ataxin-7, creating a feed forward effect that agonizes

the SCA7 disease pathway by favoring increased production of

mutant ataxin-7 protein (Figure 8). Although we cannot exclude

a role for altered transcript stability in this process, a survey of

histone posttranslational modifications in our SCA7 transgenic

mice revealed repressive chromatin modifications in the alterna-

tive promoter when SCAANT1 transcription is robust, indicating

that transcriptional activity is likely more important than tran-

script stability in controlling ataxin-7 sense expression. As bidi-

rectional transcription in association with CTCF binding is

emerging as a common feature of repeat disease loci, our find-

ings could be applicable at other repeat disease loci, including

loci that have not been carefully screened for antisense tran-

scripts. Furthermore, the existence of regulatory bidirectional

transcription may offer an entry point for therapeutically

modulating ataxin-7 expression at the RNA level. In addition to

providing a window into how gene transcription is regulated,

convergent transcription may dictate which repeat loci are

subject to dramatic genetic instability, as epigenetic modifica-

tions and chromatin alterations likely lie at the heart of the insta-

bility process. Hence, regulatory bidirectional transcription at

repeat loci, coupled with CTCF-mediated epigenetic processes

in the germline, may explain why certain repeats exhibit dramatic

parent-of-origin instability, while other repeats are relatively

stable and not subject to parent-of-origin effects (La Spada,

1997; Pearson et al., 2005). Understanding the roles and regula-

tion of convergent, bidirectional transcription at repeat loci

should provide valuable clues as to how global transcriptional

processes and epigenetic pathways work.

EXPERIMENTAL PROCEDURES

Characterization of Transgenic Mice

The generation of the SCA7 transgenic constructs, and the production of the

SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice have been previously

described (Libby et al., 2008). ERG analysis, immunostaining analysis, RT-

PCR studies, western blot analysis, and behavioral studies were performed

as previously described (Garden et al., 2002; La Spada et al., 2001). All exper-

iments were approved by the University of Washington IACUC and UCSD

IACUC. Please see Supplemental Information for detailed protocols for chro-

matin immunoprecipitation and in situ hybridization.

RNA Isolation and Analysis

Cell line, patient, and mouse tissue RNAs were isolated using Trizol and

treated 2–3 times with DNase I (NEB), then precipitated for analysis. The integ-

rity of RNA was determined by nested RT-PCR of cDNA generated with or

without reverse transcriptase, using primers to at least 3 distinct genomic

regions. For RT-PCR, cDNA was generated using gene-specific primers,

which had a linker (LK) sequence, LK 50-CGACTGGAGCACGAGGACA

CTGA-30 attached to the 50 end. Otherwise cDNA was generated with random

decamers (Ambion). cDNAwas generated with 1–3 mg of RNA and Superscript

III (Invitrogen) at 50�C. PCR amplification was performed using two gene

specific primers or with a primer to the Linker sequence and a gene-specific

reverse primer for strand-specific analysis (35 cycles at 94�C for 30 s, 55�Cfor 30 s, and 68�C for 1 min). The PCR products were cloned into the pCR4-

TOPO vector (Invitrogen) and sequenced. Strand-specific RT-PCRwas essen-


tially performed as previously described (Ladd et al., 2007). Please see the

Supplemental Information for details of construct generation.

Statistical Analysis

All data were prepared for analysis with standard spread sheet software

(Microsoft Excel). Statistical analysis was done using Microsoft Excel, Prism

4.0 (Graph Pad), or the VassarStats website (http://faculty.vassar.edu/lowry/

vassarstats.html). For ANOVA, if statistical significance (p < 0.05) was

achieved, we performed posttest analysis to account for multiple compari-

sons. The level of significance (alpha) was always set at 0.05.

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures, one movie, and Supplemental

Experimental Procedures and can be found with this article online at doi:

10.1016/j.neuron.2011.05.027.

ACKNOWLEDGMENTS

The authors wish to thank A. Smith, S. Baccam, K. Takushi, J. Huang, and

D. Possin for outstanding technical assistance, and B. Ren for critical reading

of the manuscript. This work was supported by funding from the NIH

(GM59356, EY14061, and ARRA award GM59356-09-S1 to A.R.L., and

EY01730 [Vision Research Core] to UWMC). S.M.S. is supported by the Allen

Institute for Brain Science, founded by Paul G. Allen and Jody Patton.

Accepted: May 10, 2011

Published: June 22, 2011

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ctcf regulates ataxin-7 expression through promotion of a

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